Surface plasmon resonance sensor

ABSTRACT

An SPR sensor comprising a thin conducting layer comprising at least one conductive element formed on a surface of a transparent substrate, a light source that illuminates an interface between the conducting layer and the substrate, a photosensitive surface that generates signals from light reflected from the interface, a flow cell formed with at least one flow channel having a lumen defined by a wall formed from an elastic material and from a region of the conducting layer, and at least one hollow fluid-providing flow control apparatus having a lumen and an orifice communicating with its lumen. Fluid flow is enabled between the flow channel and the lumen of the flow control apparatus by forcing an end of the flow control apparatus through the elastic material so that the orifice communicates with the flow channel lumen.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.13/751,175 filed on Jan. 28, 2013, which is a continuation of U.S.patent application Ser. No. 13/348,663 filed on Jan. 12, 2012, now U.S.Pat. No. 8,363,223, which is a continuation of U.S. patent applicationSer. No. 13/181,547 filed on Jul. 13, 2011, now U.S. Pat. No. 8,111,400,which is a division of U.S. patent application Ser. No. 12/535,707 filedon Aug. 5, 2009, now U.S. Pat. No. 7,999,942, which is a continuation ofU.S. patent application Ser. No. 12/149,158 filed on Apr. 28, 2008, nowU.S. Pat. No. 7,586,616, which is a division of U.S. patent applicationSer. No. 10/540,940 filed on Jun. 23, 2005, now U.S. Pat. No. 7,443,507,which is a National Phase of PCT Patent Application No.PCT/IL2002/001037 filed on Dec. 25, 2002. The contents of all of theabove applications are incorporated herein by reference.

FIELD OF THE INVENTION

The invention relates to surface plasmon resonance (SPR) sensors and inparticular to methods and apparatus for forming a probe layer on an SPRsensor surface, illuminating an SPR sensor surface and controllingliquid flow in an SPR sensor.

BACKGROUND OF THE INVENTION

SPR sensors generate optical signals responsive to the dielectricconstant and/or thickness, hereinafter referred to collectively as“optical properties”, of regions of a layer, hereinafter referred to asa “probe layer”, of material contiguous with a thin layer of conductingmaterial. The thin conductive layer, hereinafter an “SPR conductor”,typically has a thickness less than about 100 nm and is generally formedfrom a metal, usually silver or gold, on a surface of a transparentsubstrate such as glass. The surface on which the SPR conductor isformed is hereinafter referred to as a “sensor surface”.

Optionally, such as in a Kretschmann configuration of an SPR sensor, thesensor surface is a first surface of a prism having a triangular crosssection. Light from a suitable light source is directed into the prismthrough a second surface of the prism so that the light is incident at anon-zero angle of incidence on the SPR conductor from inside the prism.The light is linearly polarized so that it has a “p” component ofpolarization. The SPR conductor is sufficiently thin so that for anglesgreater than the critical angle of the light at the interface betweenthe prism and the SPR conductor, the evanescent field of the lightextends substantially into the probe layer. Light from the incidentlight is reflected from interface between the sensor surface and the SPRconductor, exits the prism through a third surface of the prism and isdetected by a suitable photosurface, such as for example a CCD.

For a given wavelength of the incident light, there exists a particularangle, hereinafter a “resonance angle”, greater than the critical angle,for which the evanescent field of the p polarization component of thelight resonates with a propagation mode of charge density waves ofelectrons in the SPR conductor. The charge density waves tend topropagate along the surfaces of the SPR conductor and are conventionallyreferred to as “surface plasmons”. At the resonance angle and angleswithin an “angular resonance width”, in a neighborhood of the resonanceangle, energy is coupled from the evanescent field into surfaceplasmons.

As a result of energy absorbed from the evanescent field by the surfaceplasmons, for the given wavelength, reflectance of the light as afunction of incident angle decreases substantially for angles within theangular width of the plasmon resonance and exhibits a local minimum atthe resonance angle. In addition, phase of reflected light as a functionof angle undergoes relatively rapid change for angles within the angularwidth of the plasmon resonance.

Similarly, for a given incident angle of the incident light, thereexists a particular resonance wavelength at which the incident lightresonates with a surface plasmon in the SPR conductive layer.Reflectance of the light as a function of wavelength decreasessubstantially for wavelengths within a “wavelength resonance width” ofthe surface plasmon and exhibits a local minimum at the resonancewavelength for the given angle of incidence. Phase of reflected light asa function of wavelength undergoes relatively rapid change forwavelengths within the wavelength resonance width.

The SPR resonance angle, resonance wavelength, reflectance and phasechanges that characterize a surface plasmon resonance are hereinafterreferred to as “SPR parameters”. The SPR parameters are functions of theoptical properties of the substrate (e.g. the prism glass), the SPRconductor and, because the evanescent field extends into the probelayer, of the probe layer.

In typical operation of an SPR sensor, generally either the wavelengthof light incident on the sensor surface is maintained constant and theincident angle of the light varied or the incident angle is maintainedconstant and the wavelength varied. Signals generated by thephotosurface responsive to the light reflected to the photosurface froma region of the sensor surface under either of these conditions are usedto determine a value of at least one SPR parameter for the region. Theat least one SPR parameter is used to determine a characteristic of amaterial, hereinafter a “target material”, that affects the index ofrefraction of the probe layer by interacting with the probe layer. Thetarget material is generally a liquid or a gas, i.e. a target liquid ortarget gas, that is transported along a surface of the probe layer by asuitable “flow cell”.

For example, in some applications an SPR parameter is used to identifyand assay analytes in a target liquid or gas that flows over the sensorsurface of an SPR sensor and interact with components of the probe layerto change at least one the probe layer's optical properties. In someapplications an SPR parameter is used to determine a characteristic ofan interaction, such as for example an interaction rate, betweenmaterial in a probe layer and a target material that affects the anoptical property of the probe layer. The rate of interaction determinesa rate at which the optical property of the probe layer changes andthereby a rate of change of an SPR parameter determined by the SPRsensor. The determined rate of change of the SPR parameter is used todetermine the rate of interaction.

SPR sensors and methods are generally very sensitive to changes in anoptical property of a probe layer and have proven to be useful indetecting changes in an optical property of a probe layer generated byrelatively small stimuli. An SPR probe layer may also be configured as amultianalyte “microarray” that presents on each of a relatively largeplurality of different relatively small regions, “microspots”, of asensor surface a different probe material for interaction with a targetmaterial. Thus, for example an SPR probe layer can be configured forassaying a relatively large plurality of different analytes or forcharacterizing a relatively large plurality of interactions. As aresult, SPR sensors and methods are finding increasing use inbiochemical applications and SPR sensors and methods are used toidentify and assay biomolecules and characterize reactions betweenbiomolecules.

An article by Charles E. H. Berger et al. entitled “Surface PlasmonResonance Multisensing”, Anal. Chem. Vol. 70, February 1998, pp 703-706,the disclosure of which is incorporated herein by reference, describesan SPR sensor and method that are used to characterize binding ofantigens to antibodies. The SPR sensor has a gold SPR conductor formedon a surface, i.e. a sensor surface, of a glass plate, which isoptically coupled to a prism. A flow cell comprising four parallellinear “microchannels” (generally, flow channels having at least onedimension about equal to or smaller than a millimeter), each 1 mm wide,10 mm long and about 0.1 mm deep, is positioned over the SPR conductor.A different antibody is pumped through each microchannel and adsorbed onthe gold conductor to form a probe layer. The resulting multi-analyteprobe layer comprises a linear array of four different antibodies, eachimmobilized in a different “antibody” strip on the SPR conductor.

The flow cell is then repositioned so that the microchannels areperpendicular to the antibody strips. A different antigen is pumpedthrough each of the microchannels. Each of the antigens thus comes intocontact with each of the four antibodies adsorbed onto the goldconductor. To an extent that the antigen binds with a particular one ofthe antibodies, it changes an optical property of a region of theantibody strip on which the particular antibody that contacts theantigen is located. Rates at which each antigen of the four antigensbinds to each of the four antibodies are determined from measurements ofchanges in reflectance for light incident on the sensor surface at anangle near to an SPR resonance angle. The article notes that whereas theprobe layer was formed by flowing antibodies through microchannels,other methods for forming the probe layer, such as by depositing smallquantities of antigen in specific locations using an ink jet nozzle, maybe used.

PCT publication WO 02/055993, the disclosure of which is incorporatedherein by reference, notes that “electrostatic fields can be used forcontrolling the extent of immobilization or attachment of biomolecules,such as thiol-derivitized oligonucleotides”, to a surface. The book“Microarray Analysis”, by Mark Schena, John Wiley and Sons, Inc. 2003,the disclosure of which is incorporated herein by reference, describesvarious methods for depositing or creating small quantities of desiredligands in microspots on a surface to manufacture microarrays. Among themethods described, for example in chapter seven of the book, are contactand non-contact printing methods and photolithographic methods.

U.S. Pat. No. 5,313,264, the disclosure of which is incorporated hereinby reference, describes an SPR sensor having a “liquid handling block”comprising a network of microconduits and valves. The network ofmicroconduits and valves is used for moving suitable liquids containingprobe material across an SPR conductor formed on a sensor surface so asto generate a probe layer on the SPR conductor and subsequently formoving a target liquid over the probe layer.

The SPR sensor also comprises a substantially monochromatic light sourceand an optical system for generating a wedge-shaped converging beam fromlight provided by the light source and directing the wedge-beam onto thesensor surface. The wedge-beam illuminates the probe layer along aspatially fixed, relatively narrow strip-shaped region of the sensorsurface with light that is simultaneously incident on the region in arange of incident angles. The range of incident angles is determined byan angle of convergence of the wedge-beam. Light reflected from thesensor surface is imaged on a “two dimensional photodetector device”.Signals provided by the photodetector device are processed to provide ameasurement of a change in the refractive index of the probe layer dueto interaction of material in the probe layer with material in a targetsolution that is transported along the probe layer by the liquidhandling block.

Many conventional SPR methods and apparatus for forming probe layers,flowing liquids over probe layer surfaces and optically scanning sensorsurfaces are relatively complicated, expensive and/or time consuming.Alternative SPR sensors and methods for generating multi-analyte probelayers, pumping liquids over probe layers and illuminating sensorsurfaces are needed.

SUMMARY OF THE INVENTION

An aspect of some embodiments of the present invention relates toproviding a new SPR sensor for simultaneously determining acharacteristic of each of a plurality of different interactions betweenprobe and target materials and/or identifying a plurality of differentcomponents in target materials.

An aspect of some embodiments of the present invention relates toproviding methods and apparatus for producing a microarray probe layerin an SPR sensor.

In accordance with some embodiments of the present invention, an SPRconductor in the SPR sensor comprises a plurality of conducting elementsformed on a sensor surface of a suitable substrate. Optionally, theconducting elements comprise a plurality of parallel conducting strips.A flow apparatus in the SPR sensor comprises a flow cell having aplurality of optionally parallel flow microchannels formed on a surfaceof the flow cell. The flow cell is positioned on the sensor surface sothat each microchannel crosses over each of the conducting strips andliquid flowing in each of the flow channels contacts each of the strips.Optionally, the flow channels are perpendicular to the strip conductors.A region of a strip conductor over which a flow channel crosses isreferred to as a “crossover region”.

Each of the conducting strips, hereinafter “strip electrodes”, isconnected to a power supply so that each strip electrode may beelectrified relative to a suitable reference electrode independent ofelectrification of the other of the plurality of electrodes. When astrip electrode is electrified it generates an electric field in themicrochannels. Depending upon the direction of the electric field andcharge or charge distribution carried by ligands in liquids flowingthrough the microchannels, the ligands may be attracted to or repelledfrom the electrode.

To immobilize a desired ligand on a particular crossover region of agiven electrode, a liquid containing the desired ligand is pumpedthrough the microchannel that crosses over the particular crossoverregion. The given strip electrode is electrified to attract the desiredligand to the electrode so that the ligand settles on and bonds to theelectrode at the crossover region. If it is desired to prevent theligand from accumulating at crossover regions of the other stripelectrodes that the flow channel crosses over, the other electrodes areelectrified so as to repel the ligand. By flowing appropriate ligandsthrough appropriate flow channels and electrifying strip electrodesappropriately, a microarray of substantially any pattern of immobilizedligands can be produced.

In accordance with an embodiment of the present invention subsequent tocreating the microarray, a suitable buffer solution is flushed throughthe microchannel to wash away non-immobilized ligands and targetsolutions that are to be examined by the SPR sensor are pumped throughthe microchannels. Unlike in prior art such as described in the articleby Charles E. H. Berger et al. entitled “Surface Plasmon ResonanceMultisensing” noted above, to examine target solutions with themicroarray the flow cell does not have to be reoriented relative to themicroarray after its production.

In some embodiments of the present invention, the conducting elementscomprised in the SPR conductor are relatively small conducting “pixelelectrodes”. Each flow channel crosses over at least one pixel electrodeand each pixel electrode is located under a single flow channel. Eachpixel electrode is connected to a power supply using methods known inthe art so that the power supply can electrify the pixel independentlyof electrification of the other pixel electrodes. Ligands having asuitable charge or a charge distribution comprised in liquids flowingthrough the microchannels may be attracted to or repelled from a givenpixel electrode by appropriately electrifying the pixel electrode.

An aspect of some embodiments of the present invention relates toproviding “flow apparatus” for controlling flow of liquids in an SPRsensor that provides and/or prevents liquid flow into a microchannel ata localized region of the microchannel without use of a valve at theregion. The localized region is referred to as a “flow control region”.

In accordance with an embodiment of the present invention, themicrochannel is defined by a wall, which at the flow control region, isformed from an elastic material. In some embodiments of the presentinvention the microchannel is formed in a flow cell formed from anelastic material. In some embodiments of the present invention, themicrochannel is formed in a flow cell produced from a non-elasticmaterial having an insert formed from an elastic material. The elasticinsert forms at least a portion of the wall of the microchannel locatedat the flow control region.

In accordance with an embodiment of the present invention a hollowneedle, such as for example a syringe needle, hereinafter referred to asa “flow needle”, having an orifice that communicates with the needle'slumen is used to control gas or liquid flow at the junction region. Theelastic material at the flow control region is punctured by the needleand the needle pushed into the channel so that it at least partiallyprotrudes into the channel's lumen with the needle orifice substantiallyaligned with the channel lumen. A gas or liquid fluid is pumped into oraspirated from the microchannel through the needle via the needleorifice by any of many suitable devices and methods known in the art,such as a pump or pumps.

In some embodiments of the present invention the needle, when introducedinto the microchannel lumen functions as a baffle that at leastpartially blocks fluid flow into a downstream portion of themicrochannel from an upstream portion of the microchannel or fromanother microchannel.

In accordance with an embodiment of the present invention, uponsufficient extraction of the needle from the microchannel and theelastic substrate material, the elastic material substantially seals ahole formed therein as a result of insertion of the needle into themicrochannel. As a result, a configuration of microchannel connectionsdisturbed by the insertion of the needle is returned upon extraction ofthe needle substantially to the way it was prior to the disturbance.

In some embodiments of the present invention the needle is formed with adepression, hereinafter referred to as a “shunt depression”, in theneedle's wall. Upon sufficient penetration of the needle into themicrochannel lumen, the shunt depression is substantially aligned withthe microchannel and functions as a “shunt” microchannel that connectsan upstream portion of the microchannel with another microchannel. Theshunt depression shunts flow of liquid from the upstream portion to theother channel.

It is noted that a “valveless” flow cell produced in accordance with anembodiment of the present invention is expected to be generally lessexpensive to produce than prior art flow cells comprising valves tocontrol liquid flow. As a result, a flow cell made in accordance with anembodiment of the present invention may be sufficiently inexpensive tobe disposable after being used once. By using a “disposable” flow cellonce, possibility of contamination of fluids that are pumped through theflow cell may be reduced.

An aspect of some embodiments of the present invention relates toproviding an illumination system for SPR sensors for illuminating a samerelatively large region of a sensor surface with light at a samewavelength at each of a plurality of selectable angles of incidence. Inaccordance with an embodiment of the present invention, the illuminationsystem does not require moving components to select different ones ofthe plurality of incident angles.

An aspect of some embodiments of the present invention relates toproviding an illumination system for SPR sensors for illuminating a samerelatively large region of an SPR sensor surface at a same angle ofincidence with light at each of a plurality of selectable wavelengths.In accordance with an embodiment of the present invention, theillumination system does not require moving components to selectdifferent ones of the plurality of wavelengths.

In accordance with an embodiment of the present invention, anillumination system comprises an array of light sources. An opticalsystem collimates light from any given light source in the array into abeam of substantially parallel light rays all of which are incident onthe sensor surface at substantially a same incident angle. The incidentangle is a function of the position of the light source.

In some embodiments of the present invention, for at least a subset ofthe light sources in the array, the positions of the light sources aresuch that the incident angle for different light sources is different.Each of the light sources in the at least a subset provides light at asame wavelength. The sensor surface is illuminated with light at thewavelength and different incident angles by suitably turning on andturning off light sources in the at least a subset of light sources.

In some embodiments of the present invention for at least a subset ofthe light sources the positions of the light sources are such that theincident angle for each of the light sources is substantially the same.Each of the light sources in the at least a subset provides light at adifferent wavelength. The sensor surface is illuminated with light atthe incident angle and different wavelengths by suitably turning on andturning off light sources in the at least a subset of light sources.

There is therefore provided in accordance with an embodiment of thepresent invention, an SPR sensor comprising: a thin conducting layercomprising at least one conductive element formed on a surface of atransparent substrate; an illumination system controllable to illuminatean interface between the conducting layer and the substrate; aphotosensitive surface that generates signals responsive to light fromthe light source that is reflected from a region of the interface; aflow cell formed with at least one flow channel having a lumen definedby a wall at least a portion of which is formed from an elastic materialand a portion of which is formed by a region of the conducting layer;and at least one hollow needle having an exit orifice communicating withthe needle's lumen and wherein fluid flow is enabled between the flowchannel and the needle's lumen by puncturing the elastic material withthe at least one needle so that the exit orifice communicates with theflow channel lumen.

Optionally, the flows cell is produced from of an elastic material.

In some embodiments of the present invention, the flow cell is formedfrom a relatively non-elastic material having an insert formed from anelastic material and wherein material of the insert forms at least aportion of the wall of the at least one flow channel.

In some embodiments of the present invention the end of the needle isclosed and the exit orifice is located along the length of the needle.

In some embodiments of the present invention when the needle protrudesinto the channel it at least partially blocks flow of a fluid from aportion of the channel upstream of the needle to a portion of the needledownstream of the needle.

Optionally, when the needle protrudes into the channel, the needleblocks substantially all fluid flow from the upstream portion to thedownstream portion of the channel.

In some embodiments of the present invention, the needle is formed witha depression in the needle wall and wherein when the needle protrudesinto the channel the depression forms a shunt channel between theupstream portion of the channel and another channel and at least aportion of a liquid flowing from the upstream portion of the channeltowards the downstream portion is shunted through the shunt channel tothe other channel.

In some embodiments of the present invention, upon extraction of theneedle a sufficient distance from the elastic material a hole made inthe elastic material as a result of the puncturing seals.

In some embodiments of the present invention, the at least one needlecomprises at least two needles for a channel of the at least one channeland to cause a fluid to flow in the channel both needles puncture theelastic material and are positioned to protrude into the channel withtheir respective orifices communicating with the channel lumen so thatfluid may be pumped into the channel via one of the needles andaspirated from the channel via the other of the needles. Optionally, thechannel is a blind channel having neither an inlet or outlet orifice.

In some embodiments of the present invention the SPR sensor comprises afluid pump coupled to the at least one needle controllable to pump fluidinto the needle and thereby, when the needle orifice communicates withthe flow channel lumen, into the flow channel.

In some embodiments of the present invention, the SPR sensor comprises afluid pump coupled to the at least one needle controllable to aspiratefluid from the needle and thereby, when the needle orifice communicateswith the flow channel, from the flow channel.

In some embodiments of the present invention, the illumination systemcomprises: an array of light sources; a collimator that directs lightfrom each light source in a collimated beam of light that enters thesubstrate and is incident on a region of the interface between thesubstrate and conducting layer region that forms the wall portion ofeach of the at least one flow channel; and a light source controllercontrollable to turn off and turn on a light source in the arrayindependent of the other light sources in the array.

There is further provided in accordance with an embodiment of thepresent invention, an SPR sensor comprising: a thin conducting layercomprising at least one conductive element formed on a surface of atransparent substrate; a flow cell formed with at least one flow channelhaving a lumen defined by a wall a portion of which is formed by aregion the conducting layer; a photosensitive surface that generatessignals responsive to light reflected from a region of the interfacebetween the region of the conducting layer that forms the wall portionof each of the at least one flow channel and the substrate; and anillumination system comprising: an array of light sources; a collimatorthat directs light from each light source in a collimated beam of lightthat enters the substrate and is incident on a region of the interfacebetween the substrate and conducting layer region that forms the wallportion of each of the at least one flow channel; and a light sourcecontroller controllable to turn off and turn on a light source in thearray independent of the other light sources in the array.

Additionally or alternatively, the array is a linear array having anarray axis. Optionally, the axis of the array and a normal to theinterface are substantially coplanar. Alternatively, the axis of thearray and the normal are optionally substantially perpendicular.

In some embodiments of the present invention the array is a twodimensional array. Optionally, the array comprises rows and columns oflight sources. Optionally, each column is substantially coplanar with anormal to the interface. Alternatively or additionally, each row issubstantially perpendicular to the normal.

In some embodiments of the present invention light sources in a samecolumn provide light at substantially same wavelengths.

In some embodiments of the present invention all the light sources inthe array provide light at substantially same wavelengths.

In some embodiments of the present invention, light sources in a samerow provide light at different wavelengths.

In some embodiments of the present invention the SPR sensor comprises anoptical element having two parallel surfaces through which light fromeach light sources passes before it is incident on the interface andwherein the optical element is rotatable about an axis perpendicular tothe normal so as to change an angle at which light from a given lightsource is incident on the interface.

In some embodiments of the present invention the at least one conductiveelement comprises a plurality of conductive elements.

There is further provided, in accordance with an embodiment of thepresent invention, an SPR sensor comprising: a thin conducting layercomprising a plurality of conducting elements formed on a surface of atransparent substrate; an illumination system controllable to illuminatean interface between the conducting layer and the substrate; aphotosensitive surface that generates signals responsive to light fromthe light source that is reflected from a region of the interface; and aflow cell formed with at least one flow channel having a lumen definedby a wall a portion of which is formed by a region the conducting layer.

Additionally or alternatively, each conductive element is connected to apower source controllable to electrify the conducting element withrespect to a reference electrode.

In some embodiments of the present invention, the plurality ofconductive element comprises a plurality of conducting strips.Optionally, each of the at least one flow channel crosses over eachconducting strip.

In some embodiments of the present invention, the plurality ofconductive elements comprises a plurality of conducting pixels.Optionally, each of the at least one flow channel passes over at leastone conducing pixel and each pixel lies under a flow channel.

In some embodiments of the present invention the SPR sensor comprises anexclusive reference electrode for each conducting element relative towhich the conducting element is electrified.

In some embodiments of the present invention all the conducting elementsare electrified relative to a same reference electrode.

Alternatively or additionally, the reference electrode is located on anexternal surface of the flow cell.

In some embodiments of the present invention the reference electrode islocated inside the material from which the flow cell is formed.

In some embodiments of the present invention the reference electrode islocated on the surface of the substrate. Optionally, the referenceelectrode is comb shaped having parallel conducting teeth connected to acommon backbone. Optionally, the conductive elements are located betweenthe conducting teeth.

In some embodiments of the present invention, the flow channel has across section area less than or equal to about a square millimeter.Optionally, the flow channel has a cross section area less than or equalto about 0.5 square millimeters. Optionally, the flow channel has across section area less than or equal to about 0.2 square millimeters.Optionally, the flow channel has a cross section area less than or equalto about 0.1 square millimeters.

In some embodiments of the present invention the at least one flowchannel comprises a plurality of channels.

BRIEF DESCRIPTION OF FIGURES

Non-limiting examples of embodiments of the present invention aredescribed below with reference to figures attached hereto and listedbelow. In the figures, identical structures, elements or parts thatappear in more than one figure are generally labeled with a same numeralin all the figures in which they appear. Dimensions of components andfeatures shown in the figures are chosen for convenience and clarity ofpresentation and are not necessarily shown to scale.

FIGS. 1A-1F schematically show SPR sensor, in accordance withembodiments of the present invention;

FIG. 2 schematically shows an SPR sensor with a flow needle insertingfluid into a microchannel, in accordance with an embodiment of thepresent invention;

FIGS. 3A-3D show schematic cross section views illustrating a process bywhich a flow needle punctures a flow cell in order to insert fluid intoa microchannel in the flow cell and shunt fluid flowing into themicrochannel to a drain microchannel, in accordance with an embodimentof the present invention;

FIG. 3E schematically shows a cross sectional view of a flow cell havinga microchannel and elastic inserts, in accordance with an embodiment ofthe present invention;

FIG. 4 schematically shows an SPR sensor, for performing an SPRwavelength scan of a sensor surface, in accordance with an embodiment ofthe present invention; and

FIG. 5 schematically shows an SPR sensor, for performing an SPRwavelength scan and/or an SPR incident angle scan of a sensor surface,in accordance with an embodiment of the present invention.

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

FIG. 1A schematically shows an SPR sensor 20 in accordance with anembodiment of the present invention. For convenience of discussionlocation and position of elements and features of SPR sensor 20 arereferred to a coordinate system 22.

SPR sensor 20 comprises an optionally linear array 24 of light sources26 having an array axis 27 and a prism 30 having a sensor surface 32. Aflow cell 34 comprises microchannels 36 for flowing liquid across and incontact with sensor surface 32 and a probe layer (not shown) of desiredligands generated on a suitable SPR conductor formed on the sensorsurface. In SPR sensor 20 the probe layer is generated on an SPRconductor optionally comprising a plurality of strip electrodes 33.

An array 40 of flow needles 42 coupled to suitable pumping apparatus(not shown) is optionally used to introduce liquids into microchannels36. Operation of flow needles 42 in introducing liquid intomicrochannels 36 is described below. Microchannels 36 and flow needles42 are shown having square or rectangular cross sections for convenienceof presentation and cross section shapes of the microchannels and flowneedles, in accordance with an embodiment of the present invention mayhave cross section shapes other than square or rectangular. For example,the cross section shapes may be round, oval or irregular. In addition,the sharp rectangular bends in microchannels 36 may be rounded andgradual. In some configurations of microchannels in accordance withembodiments of the present invention, bends such as though shown in FIG.1A, may not exist.

Microchannels 36 optionally have a cross section less than or equal toabout 1 sq.-mm. Optionally the microchannel has a cross section lessthan or equal to about 0.5 sq.-mm. Optionally the microchannel crosssection is less than about 0.2 sq.-mm. Optionally the microchannel crosssection is less than about 0.1 sq.-mm.

The outer form of flow cell 34 is shown in ghost lines and details ofinternal features, such as microchannels 36, of the flow cell are shownin solid lines for clarity of presentation. Sensor surface 32 isarbitrarily located in the xy-plane of coordinate system 22, lightsources 26 provide light at a same wavelength appropriate for intendedSPR angular scan applications and array axis 27 of linear array 24optionally lies in the yz-plane.

Linear array 24 is positioned at the focal plane of an optical systemschematically represented by a lens 46 having an optical axis 48 in theyz-plane. Lens 46 collects and collimates light from each light source26 into a beam of parallel light rays and directs the collimated lightso that it is incident on an “input” prism surface 50 of prism 30. Anormal to input surface 50 is optionally parallel to the yz-plane. Lightdirected by collimator 46 that is incident on input surface 50 entersprism 30 and is incident on sensor surface 32.

All light incident on sensor surface 32 from a given light source 26 isincident on the sensor surface at substantially a same incident angleand light from different light sources 26 is incident on the sensorsurface at different incident angles. The angle at which light from agiven light source 26 is incident on sensor surface 32 is determined bythe position of the given light source along the axis of linear array24, the focal length “f” of lens 46 and the index of refraction “n” ofmaterial from which prism 30 is formed. An angular difference betweenthe angles of incidence on sensor surface 32 of light from two adjacentlight sources 26 is approximately equal to (D/f)(1/n), where D is adistance between the adjacent light sources 26. Optionally, distance Dbetween any two adjacent light sources 26 along array axis 27 is thesame.

It is noted that incident angles available from light source array 24are “quantized” in steps of (D/f)(1/n) radians. In some embodiments ofthe present invention, an SPR sensor such as SPR sensor 21 shown in FIG.1B, comprises a “displacement plate” 100 formed from a suitablytransparent material and having parallel surfaces 101 and 102 ispositioned between light source array 24 and prism 30. Except fordisplacement plate 100, SPR sensor 21 is identical to SPR sensor 20.Displacement plate 100 is optionally positioned between light sourcearray 24 and lens 46 and is controllable to be rotated about an axis 104parallel to the x-axis. Angular orientation of displacement plate 100 isthereby controllable so that a normal (not shown) to surfaces 101 and102 can be oriented at a desired angle with respect to optic axis 48.

For non-zero “displacement angles” between optic axis 48 and the normalto surfaces 101 and 102, displacement plate 100 generates virtual imagesof light sources 26 that are displaced along array axis 27. Magnitude ofdisplacement of light sources 26 is a function of the displacementangle, distance between surfaces 101 and 102 and index of refraction ofthe material from which displacement plate 100 is formed. By suitablyrotating displacement plate 100, light from any light source 26 can bedirected to be incident on sensor surface 32 at substantially anydesired incident angle and not only at a quantized incident angle.

Light incident on sensor surface 32 that is reflected from the surfaceexits prism 30 through an output prism surface 52 and is collected andimaged by a suitable optical system represented by a lens 53 onto a twodimensional photosurface 54 such as a CCD. A polarizer (not shown) ispositioned between array 24 and prism 30 or preferably between prism 30and photosurface 54. The polarizer linearly polarizes light received byphotosurface 54 so that relative to sensor surface 32 it hassubstantially only a p component of polarization.

Whereas the SPR conductor shown in FIG. 1A (and FIG. 1B, however FIG. 1Awill generally be used as reference for features common to SPR sensors21 and 22) has only five strip electrodes 33, the number is by way ofexample and a number of strip electrodes other than five may be used inthe practice of the present invention. For example, in some embodimentsof the present invention strip electrodes 33 cover a region of sensorsurface 32 having an extent in the x and the y directions equal to about20 mm. Each strip electrode 33 has a width of, optionally, about 100micrometers and the electrodes are optionally formed on sensor surface32 at a pitch of about 200 micrometers. For these dimensions the numberof strip electrodes 33 on sensor surface 32 is about 100.

Microchannels 36 in flow cell 34 are optionally parallel and flow cell34 is mounted to prism 30 so that the microchannels are optionallyperpendicular to strip-electrodes 33. Each microchannel 36 optionallyhas an inlet segment 55 and a segment 56 that is open on a side of themicrochannel facing sensor surface 32 so that fluid flowing in themicrochannel contacts each strip electrode 33 that the microchannelcrosses at a crossover region 58. Regions of some microchannels 36 inSPR sensor 20 in FIG. 1A are cut away to show crossover regions 58. Eachmicrochannel 36 optionally has an open ended outlet segment 61 throughwhich fluid flowing in the microchannel may exit the microchannel.

In accordance with an embodiment of the present invention, each stripelectrode 33 is connected to a power supply 60. Power supply 60 iscontrollable to electrify each strip electrode 33 relative to a suitablereference electrode connected to the power supply so as to generate anelectric field having a component perpendicular to sensor surface 32 ateach of the electrode's cross over regions 58. The electric field ateach cross over region passes through the lumen of the microchannel 36that crosses over the electrode at the crossover region. Toappropriately electrically isolate each strip electrode 33, flow cell 34is formed from an insulating material or is appropriately covered withan insulating material. For convenience of presentation it is assumedhereinafter that flow cell 34 and other flow cells, in accordance withan embodiment of the present invention, are formed from a suitableinsulating material although parts of the flow cell may be formed from aconducting material.

In some embodiments of the present invention, as shown for SPR sensor 20in FIG. 1A, each strip electrode 33 is electrified relative to a samerelatively large reference electrode 62 located on a top surface 64 offlow cell 34. In some embodiments of the present invention, referenceelectrode 62 is “buried” in flow cell 34 so as to bring each stripelectrode closer to the large electrode. Bringing reference electrode 62closer to strip electrodes 33 tends to concentrate the electric fieldbetween an electrified strip electrode 33 and the reference electrodewithin a volume of space sandwiched between the strip electrode and thereference electrode and thereby reduce “cross-talk” between stripelectrodes.

In some embodiments of the present invention top surface 64 has arecessed portion relatively closer to strip electrodes 33 than otherregions of the top surface. Reference electrode 62 is mounted to therecessed portion so as to reduce distance between the referenceelectrode and strip-electrodes 33. FIG. 1C schematically shows an SPRsensor 70, in accordance with an embodiment of the present inventionsimilar to SPR sensor in which a top surface 72 of a flow cell 74 has arecessed portion 75 on which a reference electrode 62 is mounted. Forclarity of presentation in FIG. 1C internal microchannels, otherinternal features of flow cell 74 and strip electrodes 33 are not shown.

In some embodiments of the present invention each strip electrode 33 hasits own exclusive “partner” reference electrode relative to which thestrip electrode is electrified by power supply 60. Such a partnerelectrode is optionally a mirror image of the strip electrode to whichit is a partner. Optionally, each strip electrode's partner electrode isburied inside flow cell 34. FIG. 1D schematically shows an SPR sensor 80having a flow cell 82, in accordance with an embodiment of the presentinvention, in which each strip electrode 33 has its own mirror imagepartner electrode 83 buried in the flow cell.

In some embodiments of the present invention an SPR conductor on sensorsurface 32 comprises a plurality of pixel electrodes instead ofstrip-electrodes 33. FIG. 1E schematically shows an SPR sensor 140comprising an SPR conductor having pixels electrodes 142. For clarity ofpresentation internal features of flow cell 34 are not shown in FIG. 1Eand the flow cell and reference electrode 62 are shown in ghost lines.Pixel electrodes 142 are arrayed in optionally parallel rows 144, eachof which is optionally perpendicular to microchannels 36 (FIG. 1A) inflow cell 34 and each flow cell in a row 144 is located under adifferent microchannel. Each pixel electrode 142 is connected to powersupply 60 and may be electrified relative to reference electrode 62independent of electrification of the other pixel electrodes.

In some embodiments of the present invention both an SPR conductor and areference conductor or conductors are located on an SPR sensor surface.By way of example, FIG. 1F schematically shows an SPR sensor 150, inaccordance with an embodiment of the present invention having an SPRconductor comprising a plurality of strip electrodes 152 and a referenceelectrode 154 both of which are located on the SPR sensor's sensorsurface 32. Reference electrode 154 is in the form of a comb havingteeth 156 that interleave with strip electrodes 152 and is a referenceelectrode common to all the strip electrodes. Optionally referenceelectrode 152 is grounded. Each strip electrode 152 is electrified bypower supply 60 relative to reference electrode 154 independent of theelectrification of other of the strip electrodes.

In accordance with an embodiment of the present invention, a flow cellis formed from an elastic material and liquids are introduced into amicrochannel formed in the flow cell by puncturing the elastic materialwith a flow needle until an outlet orifice of the flow needle issubstantially aligned with the microchannel. Liquid is pumped into themicrochannel from the flow needle's lumen to the microchannel via theorifice. Any of various methods and “positioning” apparatus known in theart may be used to control movement and positioning of the flow needlesand controlling liquid flow into and out of the flow needles. A methodof controlling fluid flow in microchannels of a flow cell using flowneedles, in accordance with an embodiment of the present invention isdiscussed with reference to FIG. 1A.

Liquids are introduced into microchannels 36 of flow cell 34 shown inFIG. 1A either through their respective inlet segments 55 or byinjection through flow needles 42. Each microchannel 36 is associatedwith its own flow needle 42 and position of the flow needle determineswhether liquid from inlet segment 55 or from flow needle 42 flows in themicrochannel. Each microchannel 36 is also associated with its own drainmicrochannel 59. A microchannel 36 and its drain microchannel 59 are notconnected by a flow channel formed in the flow cell 34.

Each flow needle 42 has an outlet orifice 43 optionally located alongthe length of the flow needle that communicates with the flow needle'slumen and an optionally closed, relatively sharp tip 45. Optionally, adepression 47, i.e. a “shunt depression 47” is formed on a “back-side”wall 49 of flow needle 42 opposite its outlet orifice 43. Inset 90 inFIG. 1 shows a schematic enlarged view of a flow needle 42 that showsback-side wall 49 of the flow needle and its shunt depression 47.

Whereas in FIG. 1A shunt depression 47 has a width less than a width ofbackside wall 49 in some embodiments of the present invention, shuntdepression 47 has a width substantially equal to that of backside wall49 and such a width can be advantageous. It is noted that whereas flowneedles 42 have their respective orifices 43 located along their lengthsand are shown with closed ends, flow needles suitable for the practiceof the present invention may have open ends and these open ends mayfunction also as exit orifices.

In accordance with an embodiment of the present invention, a flow needle42 has an extracted position and an inserted position. In FIG. 1A allflow needles 42 are shown in the extracted position. In the extractedposition a flow needle 42 does not affect fluid flow in its associatedmicrochannel 36 and liquid pumped into inlet segment 55 of theassociated microchannel will flow in the microchannel.

To move flow needle 42 from its extracted position to its insertedposition, in accordance with an embodiment of the present invention, theflow needle is forced into flow cell 34 so that it cuts through andpenetrates the elastic flow cell material. The flow needle is inserteduntil the flow needle's orifice 43 is substantially aligned with thelumen of its associated microchannel 36. In the inserted position flowneedle 42 blocks liquid flow from inlet segment 55 of microchannel 36into the microchannel's lumen downstream of the inlet segment andenables flow of liquid from the flow needle's lumen into themicrochannel. For those flow needles 42, which in accordance with anembodiment of the present invention have a shunt depression, in theinserted position the flow needle shunts liquid pumped into inletsegment 55 of microchannel 36 to the microchannel's drain microchannel59.

FIG. 2 schematically shows SPR sensor 20 with a left-most flow needle42, which is individualized by the numeral 92, in an inserted position.FIGS. 3A-3D schematically illustrate cross sectional views of flowneedle 92 being moved from its extracted position to its insertedposition in flow cell 34. In the cross-sectional views, orifice 43 offlow needle 92 is indicated by a gap in the wall of the flow needle andshunt depression 47 as a recess in the wall.

In FIG. 3A flow needle 92 is in the extracted position and liquid,indicated by arrowhead lines 94 is being pumped into inlet segment 55 ofits associated microchannel 36 from a suitable source. Liquid 94 flowsfreely from inlet segment 55 into and through microchannel 36. In FIG.3B flow needle 92 is lowered into drain microchannel 59 until its tip 45is touching a region 96, hereinafter referred to as a “septum 96”, offlow cell 34 that separates drain microchannel 59 from microchannel 36.In FIG. 3C flow needle 92 is schematically being forced through septum96.

In FIG. 3D, flow needle 92 has penetrated flow cell 34 sufficiently sothat its outlet orifice 43 is substantially aligned with microchannel 36and the flow needle is in its inserted position. In addition, in theinserted position, shunt depression 47 is substantially aligned to forma shunt flow channel between inlet segment 55 and drain microchannel 59.Shunt depression 47 is sufficiently deep and narrow so that the elasticmaterial of flow cell 34 does not squeeze into the shunt depression andseal it. Liquid represented by arrowhead lines 98 flows from flow needle92 through the flow needle's exit orifice 43 into microchannel 36. Flowof liquid 94 from inlet segment 55 into a portion of microchannel 36downstream of the flow needle is substantially blocked by the flowneedle and is shunted via shunt depression 47 to drain microchannel 59,from which drain microchannel the liquid exits flow cell 34.

In some embodiments of the present invention to provide for a degree ofplay in alignment of exit orifice 43 with microchannel 36 when flowneedle 92 is in the inserted position, microchannel 36 has a relativelyenlarged cross section in a region of the microchannel in which the flowneedle is introduced. Alternatively or additionally exit orifice 43 maybe smaller than the microchannel cross section in the region of themicrochannel in which flow needle 92 is introduced.

Whereas in the above example flow cell 34 is assumed to be formed froman elastic material, in some embodiments of the present invention, aflow cell is formed from a relatively inelastic material. To provideregions of a microchannel for which a flow needle can be introduced intothe microchannel, in accordance with an embodiment of the presentinvention, the flow cell comprises elastic inserts, which form regionsof the microchannel. A flow needle may positioned in the flow channel,in accordance with an embodiment of the present invention by suitablepuncturing the elastic inserts.

FIG. 3E schematically shows a cross sectional view of a flow cell 160having a microchannel 162 formed therein, in accordance with anembodiment of the present invention. Flow cell 160 is produced from arelatively inelastic material and to provide a suitable region throughwhich to introduce a flow needle into microchannel 162, in accordancewith an embodiment of the resent invention, the flow cell is fitted withelastic inserts 164 and 166. A channel 59 has a portion thereof formedin the relatively inelastic material of flow cell 160 and a portionthereof formed in elastic insert 164.

It is noted that in FIGS. 3A-3E it is assumed that fluid introduced intoa microchannel 36 by flow needle 92 exits the flow channel through openended exit segment 61. In some embodiments of the present inventionfluid introduced into a microchannel via a first flow needle may exitthe flow channel via a second flow needle rather than through an exitsegment. Both the first and second flow needles puncture regions of thewall of the microchannel formed form an elastic material and areintroduced into the lumen of the microchannel so that their respectiveorifices communicate with the lumen. The orifice of the first flowneedle faces downstream and the orifice of the second flow needle facesupstream. Fluid is introduced into the microchannel via the firstneedle, for example by pumping the fluid into the microchannel via thefirst needle's lumen. The fluid exits the microchannel via the secondflow needle, for example by aspirating the fluid from the microchannelvia the second needle's lumen.

It is further noted that in the discussion of FIGS. 1A-3E when a flowneedle 42 (FIGS. 1A-2) or individualized flow needle 92 (FIGS. 3A-3D) isintroduced into microchannel 36 it completely blocks fluid flow into themicrochannel from upstream of the needle. In some embodiments of thepresent invention a flow needle may only partially block fluid flow fromupstream of the needle. For example, the orifice of the flow needle maybe positioned so that the needle may the orifice communicates with themicrochannel lumen when the flow needle is only partially introducedinto the microchannel lumen so that it only partially blocks fluid flowfrom upstream. Alternatively the flow needle may be narrower than awidth of the cross section of the microchannel in a region of themicrochannel in which the flow needle is introduced into themicrochannel. As a result, even when fully introduced into themicrochannel a fluid from upstream may stream downstream around theneedle. A flow needle and microchannel configuration that enables theflow needle to only partially block fluid flow in the microchannel canbe advantageous when it is desired to mix a fluid introduced into themicrochannel via the flow needle with fluid flowing downstream fromupstream of the needle. Variations of the methods described for usingflow needles, in accordance with an embodiment of the present inventionto introduce and remove fluid from a microchannel will occur to a personof the art.

To illustrate operation of SPR sensor 20, in accordance with anembodiment of the present invention, assume that it is required todetermine the kinetics of interaction between a plurality of different“probe” proteins with a particular “target” proteins. By way of example,assume that the number of the plurality of probe proteins is equal tothe number (twenty five) of crossover regions 58 between microchannels36 and strip electrodes 33 in SPR sensor 20 and that a different probeprotein is to be immobilized at each crossover region. Immobilization atcross over region, in accordance with an embodiment of the presentinvention, may be made directly to the conductor from which stripelectrodes 33 are formed or to a suitable molecular layer formed on theconductor using any of various methods known in the art.

To prepare an appropriate microarray of the probe proteins on stripelectrodes 36, initially, buffer or water is pumped throughmicrochannels 36 via inlet segments 55 to clean and prepare the stripelectrodes for immobilization of the probe proteins at crossover regions58. Each flow needle 42 is aspirated, using any of various differentmethods and apparatus known in the art, with an appropriate solutioncomprising a different one of the plurality of probe proteins. Assumethat the different probe proteins are to be immobilized on a first oneof strip electrodes 33. The first strip electrode is electrifiedpositive or negative with respect to reference electrode 62 dependingupon whether the probe proteins are negatively or positively chargedrespectively. The remaining strip electrodes are all electrified withrespect to electrode 62 to voltage or voltages having polarity oppositeto polarity of a voltage to which the first electrode is electrified.Flow needles 42 with their respective nucleotide solutions arecontrolled to puncture flow cell 34 so that they are positioned in theirinserted positions.

Upon insertion of flow needles 42 to their inserted positions, flow ofbuffer or water through the microchannels via their respective inputsegments 55 is halted and buffer or water pumped the input segments isshunted to corresponding drain microchannel 59 via the flow needle'sshunt depressions 47. The probe protein solution in each flow needle 42is pumped out of the flow needle and into its associated microchannel36. As a result of the electrification pattern of strip electrodes 33and the charge on the probe protein in the solution, the probe proteinis attracted to the first strip electrode 33 and repelled by the otherstrip electrodes 33. The probe protein is thereby immobilized at the atthe crossover region 58 of the associated microchannel 33 and the firststrip electrode 33 and is substantially prevented from immobilizing atcrossover regions 58 of the other strip electrodes 33.

During immobilization of the probe proteins, the process ofimmobilization and quantities of probe proteins immobilized at crossoverregions 58 is monitored by performing an SPR angular scan of sensorsurface 64. Light sources 26 in array 24 are sequentially turned on andturned off to perform the angular SPR scan of sensor surface 64 andilluminate substantially a same region of sensor surface 64, whichincludes at least all of crossover regions 58, at a plurality ofdifferent incident angles.

Signals generated by CCD 54 responsive to light from each light source26 reflected at each crossover region 58 (i.e. from a region of sensorsurface 64 on which the crossover region is located) of the first stripelectrode 33 are used to determine an SPR parameter for the crossoverregion. The SPR parameter is used to monitor accretion of immobilizedprobe protein at the crossover region. Signals from crossover regions 58of other strip electrodes 33 and from regions of strip electrodes 33that are not crossover regions are used to correct and normalize signalsfrom crossover regions 58 of the first strip electrode 33.

Flow needles 42 are then extracted from flow cell 34. Upon extractionblockage of inlet segments 55 of microchannels 36 by flow needles 42 isremoved and “insertion holes” formed in the elastic material from whichflow cell 34 is formed due to insertion of flow needles 42 seal. Flow ofbuffer or water through microchannels 36 via inlet segments 55 resumesand purges probe proteins at crossover regions of the first stripelectrode 33 and other strip electrodes 33 and in microchannels 33 thatwere not immobilized.

The above-described process is repeated for each of the other stripelectrode 33 with solutions containing different probe proteins from theplurality of probe proteins until a different desired one of the probeproteins is immobilized at each of crossover regions 58 and the desiredmicroarray of twenty-five probe proteins is prepared.

Following preparation of the microarray, each of flow needles 42 isaspirated with a solution of the particular target protein whoseinteraction kinetics with the probe proteins is to be tested. The flowneedles are inserted into flow cell 34 to their respective insertedpositions to block flow of water or buffer from inlet segments 55 andflush each microchannel 36 with the target protein solutions. An angularSPR scan of sensor surface 62 is performed by appropriately turning onand off light sources 26. Signals provided by CCD 54 responsive to lightfrom the light sources reflected from each crossover region 58 areprocessed to monitor the interaction kinetics between the target proteinand the probe protein immobilized at the crossover region.

In the above example, interaction kinetics of a single target proteinwith each of twenty five probe proteins is monitored by SPR sensor 20.It is of course possible, in accordance with an embodiment of thepresent invention, to flow a different target protein through eachmicrochannel after preparation of the microarray. In that caseinteraction kinetics of each of five target proteins is monitored foreach of five different probe proteins. Interaction kinetics of a giventarget protein is monitored for probe proteins that are immobilized atcrossover regions between each of strip electrodes 33 and a particularmicrochannel through which the given target protein flows.

It is further noted that in describing preparation of the above notedmicroarray of twenty five probe proteins, it was tacitly assumed that inorder to configure electrification of strip electrodes 33 whenimmobilizing the proteins, all proteins pumped through microchannels 36at a same time carry a same polarity charge. Therefore, for theresulting microarray probe proteins immobilized on crossover regions 58of a same given strip electrode 33 carry a same polarity charge.However, in some embodiments of the present invention biomoleculeshaving different polarity charges are immobilized on a same given stripelectrode 33.

In general, biomolecules bound to a strip electrode 33, for example bycovalent bonds, are bound by electrical fields that are substantiallystronger than electric fields used to attract or repel biomolecules thatare generated by electrifying the strip electrode. As a result, it ispossible to bind biomolecules having opposite polarity charge to a sameelectrode strip 33.

For example, assume a first fluid comprising first biomolecules having afirst polarity charge are pumped through a flow channel 36 so as tocontact a given strip electrode 33 at a first cross over region 58. Toattract and immobilize the first biomolecules on the first crossoverregion 58, the given strip electrode is appropriately electrified toattract the first biomolecules. Subsequently a second fluid comprisingsecond biomolecules having a second polarity charge is pumped through adifferent flow channel 36 so as to contact the given strip electrode 33at second crossover region 58. To attract and immobilize the secondbiomolecules to the second crossover region 58 of strip electrode 33polarity of electrification of the strip electrode is reversed. Theelectric field generated by the reversed polarity electrification of thegiven strip electrode 33, while sufficient to attract the secondbiomolecules to the strip electrode, is not strong enough to sunderbonds between the first biomolecules, which are already bound to thestrip electrode, and the strip electrode.

It is noted that an SPR electrode comprising a plurality of pixelelectrodes, in accordance with an embodiment of the present invention,such as the SPR electrode comprising pixel electrodes 142 shown in FIG.1E, may be advantageous in preparing a microarray of biomolecules havingdifferent polarity charge configurations. A “pixelated” SPR electrode,in accordance with an embodiment of the present invention, providesincreased flexibility for generating different polarity electric fieldsat different regions of the SPR electrode.

In the above description of exemplary SPR sensors, in accordance withembodiments of the present invention, only angular SPR scans are used tomonitor processes occurring at a microarray prepared on an SPR electrodeformed on sensor surface 32. Some SPR sensors, in accordance withembodiments of the present invention, are configured to providewavelength scans of a sensor surface and comprise a light source arraythat provides light at a same incident angle and different wavelengths.

FIG. 4 schematically shows an SPR sensor 120, in accordance with anembodiment of the present invention, configured to provide SPRwavelength scans of sensor surface 32 at a constant incident angle. SPRsensor 120 is similar to SPR sensor 20 shown in FIG. 1A. However, unlikeSPR sensor 20, SPR sensor 120 optionally comprises a linear light array122 of light sources 124 for which each light source provides light at adifferent desired wavelength and an array axis 126 of the array isparallel to the x-axis.

Incident angle of light from a light source, such as a light source 26in array 24 of SPR sensor 20 (FIG. 1A) or a light source 124, isdetermined substantially only by an elevation angle of the light sourceposition measured with respect to the z-axis. The incident angle is asecond order function of an azimuth angle, as measured for example fromthe x-axis and in the xy-plane, of a light source 26 or a light source124.

Whereas each light source 26 in SPR sensor 20 (FIG. 1A) is located at asame azimuth angle (all light sources 26 are located substantially inthe yz-plane) but at a substantially different declination angle, lightsources 124 in array 120 are located at a substantially same declinationangle but substantially different azimuth angles. Different lightsources 26 therefore provide, in accordance with an embodiment of thepresent invention, light at different incident angles and are suitablefor providing angular SPR scans of SPR sensor surface 32 at constantwavelength. Light sources 124 on the other hand provide light atsubstantially a same incident angle but at different wavelengths andlight source array 122 is therefore suitable for providing an SPRwavelength scan of sensor surface 32 at a constant incident angle.

In accordance with some embodiments of the present invention, an SPRsensor comprises a two dimensional, optionally planar, array of lightsources. In some embodiments of the present invention, light sources areconfigured in the array so that they provide light at differentwavelengths and from a range of elevation angles and a range of azimuthangles. By appropriately turning on and off light sources in the array,both SPR angular and wavelength scans of an SPR sensor surface can beprovided.

FIG. 5 schematically shows an SPR sensor 130 comprising a twodimensional array 132 of light sources 134. Array 132 is optionally arectangular array and comprises rows 136 and columns 138 of lightsources 134. Rows 136 are parallel to the x-axis and each light source134 in a row 136 optionally provides light at a different wavelengthsuitable for desired SPR wavelength scans of sensor surface 32.Optionally, all light sources in a same given column 138 provide lightat a same wavelength. Light sources 134 in the column 138 are suitablefor providing an SPR angular scan of sensor surface 32 at the wavelengthof light provided by the light sources in the column.

It is noted that for performing an angular SPR scan it can be difficultto provide a light source that provides strong intensity light forilluminating an SPR sensor surface at each of a plurality of differentdesired incident angles. In some embodiments of the present inventionall light sources 134 in array 132 provide light at a same wavelength.For a configuration in which all light sources 134 provide light at asame wavelength light source array 132 may be used to perform an angularSPR scan with relatively intense light at each incident angle used inthe scan.

For example, as noted above, each light source 134 in a row 136 of array132 provides light at a same incident angle, which is definedsubstantially by the row's elevation angle. Light sources 134 indifferent rows 136 provide light at different incident angles. Assumethat the different desired scan angles for an SPR angular scan are thedifferent incident angles provided by light sources 134 in the differentrows 136. Relatively intense light may be provided at a given desiredscan incident angle by simultaneously turning on all light sources 134in the row 136 for which the light sources provide light at the givenincident angle.

As in the case of light source array 24 (as shown in FIG. 1B), lightsource array 122 and a two dimensional light source array, such as array132 shown in FIG. 5, may be combined with a displacement plate (FIG. 1B)that functions to adjust angles at which light from light sources in thearray are incident on sensor surface 32.

Whereas, aspects and features of the present invention have beendescribed as comprised in SPR sensors, the aspects and features are notlimited to use in SPR sensors. For example, illumination systems, flowapparatus and electrode configurations in accordance with embodiments ofthe present invention may be used in critical angle refractometrysystems and total internal reflection fluorescence or phosphorescencesystems.

In the description and claims of the present application, each of theverbs, “comprise” “include” and “have”, and conjugates thereof, are usedto indicate that the object or objects of the verb are not necessarily acomplete listing of members, components, elements or parts of thesubject or subjects of the verb.

The present invention has been described using detailed descriptions ofembodiments thereof that are provided by way of example and are notintended to limit the scope of the invention. The described embodimentscomprise different features, not all of which are required in allembodiments of the invention. Some embodiments of the present inventionutilize only some of the features or possible combinations of thefeatures. Variations of embodiments of the present invention that aredescribed and embodiments of the present invention comprising differentcombinations of features noted in the described embodiments will occurto persons of the art. The scope of the invention is limited only by thefollowing claims.

1. A surface plasmon resonance (SPR) system using a ligand, the systemcomprising: a) a thin conducting layer suitable for SPR formed on atransparent substrate, the conducting layer comprising a plurality ofelectrodes; b) at least one flow channel crossing the electrodes atcrossover regions; c) a power supply connected to the electrodes, anddifferently electrifying the electrodes at two different cross-overregions, such that the ligand flowing through the flow channel isattracted to and immobilized on the electrode at one of the cross-overregions, and repelled by and substantially prevented from immobilizingon the electrode at the other of the cross-over regions; and d) aphotosensitive surface that generates SPR signals in response to lightreflected from the crossover region at which the ligand is immobilized,that can be used to determine an SPR parameter, and generates referenceSPR signals in response to light reflected from the crossover region atwhich the ligand is prevented from immobilizing, that can be used tonormalize and correct the SPR signals from the crossover region at whichthe ligand is immobilized.
 2. A system according to claim 1, wherein theat least one flow channel comprises a plurality of flow channels.
 3. Asystem according to claim 2, wherein the electrodes comprise a stripelectrode crossed by the plurality of flow channels.
 4. A systemaccording to claim 2, wherein the electrodes comprise a pixel electrodecrossed by only one of the flow channels.
 5. A method for characterizingan interaction of a target material with a ligand on an SPR surface, themethod comprising: a) electrifying a first electrode comprising aconducting layer suitable for SPR formed on a transparent substrate, sothat it attracts the ligand; b) electrifying a second electrodedifferently from the first electrode, so that it repels the ligand, thesecond electrode also comprising a conducting layer suitable for SPRformed on a transparent substrate; c) passing a fluid containing theligand through a flow channel crossed by the first and second electrodeswhile electrifying them, immobilizing the ligand on the first electrodeand substantially not on the second electrode; d) passing the targetmaterial through the flow channel after the ligand has been immobilized;e) detecting light reflected from the first and second electrodes, afterpassing at least some of the target material through the flow channel;f) generating SPR signals from the detected light reflected from thefirst electrode, and SPR reference signals from the detected lightreflected from the second electrode; and g) determining an SPR parameterthat characterizes the interaction from the SPR signals, normalized andcorrected by the SPR reference signals.
 6. A method according to claim5, also comprising: a) electrifying a third electrode, comprising aconducting layer suitable for SPR formed on a transparent substrate,that also crosses the fluid channel, after the ligand is immobilized onthe first electrode, while passing fluid containing a second ligandthrough the flow channel, so that the second ligand is attracted to andimmobilized on the third electrode, and electrifying the first andsecond electrodes differently so that the second ligand is repelled byand substantially not immobilized on the first and second electrodes; b)detecting light reflected from the third electrode, after passing atleast some of the target material through the flow channel; c)generating additional SPR signals from the detected light reflected fromthe third electrode; and d) determining an SPR parameter characterizingan interaction of the target material with the second ligand from theadditional SPR signals.
 7. A system according to claim 3, wherein thestrip electrode is about 100 μm in width.
 8. A system according to claim3, wherein the electrodes comprise a plurality of substantially parallelstrip electrodes repeating at a pitch of about 200 μm.
 9. A systemaccording to claim 3, wherein the electrodes comprise about 100substantially parallel strip electrodes.
 10. A method according to claim5, wherein the first electrode is a strip electrode crossed also by asecond flow channel, the method also comprising: a) passing a fluidcontaining a second ligand, which has a polarity charge of a same signas the first ligand, through the second flow channel while the firstelectrode is electrified so that it attracts the first and secondligands, immobilizing the second ligand on the first electrode where itis crossed by the second flow channel; b) passing a second targetmaterial, the same as or different from the first target material,through the second flow channel after the second ligand has beenimmobilized; c) detecting light reflected from the first electrode whereit is crossed by the second flow channel, after passing at least some ofthe second target material through the second flow channel; d)generating second SPR signals from the detected light reflected from thefirst electrode where it is crossed by the second flow channel; and e)determining a second SPR parameter that characterizes an interaction ofthe second target material with the second ligand, from the SPR signals.11. A method according to claim 10, wherein the second electrode is alsoa strip electrode crossed also by the second flow channel, the secondligand being repelled by the second electrode and substantially notimmobilized on it when it is electrified differently from the firstelectrode, and the method also comprises detecting light reflected fromthe second electrode where it is crossed by the second flow channel andgenerating second SPR reference signals from said detected light, anddetermining a second SPR parameter that characterizes the interaction ofthe second target material with the second ligand comprises normalizingand correcting the second SPR parameter from the second referencesignals.
 12. A method according to claim 5, wherein the first electrodeis a strip electrode crossed also by a second flow channel, the methodalso comprising: a) electrifying the first electrode so it repels thefirst ligand, at a different time than when the first electrode iselectrified so it attracts the first ligand; b) passing a fluidcontaining a second ligand, which has a polarity charge of an oppositesign to the first ligand, through the second flow channel while thefirst electrode is electrified so that it repels the first ligand andattracts the second ligand, immobilizing the second ligand on the firstelectrode where it is crossed by the second flow channel; c) passing asecond target material, the same as or different from the first targetmaterial, through the second flow channel after the second ligand hasbeen immobilized; d) detecting light reflected from the first electrodewhere it is crossed by the second flow channel, after passing at leastsome of the second target material through the second flow channel; e)generating second SPR signals from the detected light reflected from thefirst electrode where it is crossed by the second flow channel; and f)determining a second SPR parameter that characterizes an interaction ofthe second target material with the second ligand, from the SPR signals.13. A method according to claim 12, wherein, after one of the twoligands is immobilized on the first electrode, and it is electrified soit repels the already immobilized ligand and attracts the other ligand,the first electrode is not electrified so strongly that the alreadyimmobilized ligand is removed from it.
 14. A surface plasmon resonance(SPR) system comprising: a) a thin conducting layer suitable for SPRformed on a transparent substrate, the conducting layer comprising anumber of conducting strips about 100 μm in width; b) a plurality offlow channels crossing the conducting strips at crossover regions; c) anillumination system configured to illuminate an interface of theconducting layer and the substrate at the crossover regions, and atother regions of the conducting strips that are not crossover regions,at a plurality of different angles of incidence or differentwavelengths; and d) a photosensitive surface that generates SPR signalsin response to light reflected from the illuminated crossover regionsthat can be used to determine an SPR parameter, and generates referenceSPR signals in response to light reflected from the other regions, thatcan be used to normalize or correct the SPR signals from the crossoverregions.